While Fibronectin (FN) is abundant and has pleiotropic functions during skeletal muscle regeneration, it remains unclear how its spatiotemporal specificity for skeletal muscle stem cells (MuSCs) is established. We discovered that activated MuSCs secrete an autoregulatory FN splice variant containing the EDB extra domain (EDB(+) FN), which is not expressed by accessory cells in the niche. In order to study the consequences of reduced levels of FN isoforms in MuSC-derived primary mouse myoblasts, we performed a proteomic analysis comparing cells treated with siRNAs targeting either (I) EDB(+) FN, (II) EDB(-) FN (targeting all FN transcripts without the EDB extra domain), or (III) siSCR (scrambled siRNA).